Stabilization of Paclitaxel-Conjugated Micelles by Cross-Linking with

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Increasing crosslinking density of core-crosslinked paclitaxel conjugated micelles compromises the antitumor effects against 2D and 3D tumor cellular models Alice W. Du, Hongxu Lu, and Martina Heide Stenzel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00410 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 25, 2016

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Molecular Pharmaceutics

Stabilisation of paclitaxel conjugated micelles by crosslinking with cystamine compromises the anti-tumor effects against 2D and 3D tumor cellular models Alice Wei Dua, Hongxu Lub, Martina Stenzelb* Centre for Advanced Macromolecular Design (CAMD), aSchool of Chemical Engineering and bSchool of Chemistry, University of New South Wales, Australia Abstract:

Paclitaxel (PTX) conjugated micelles provide a promising tool for the treatment of prostate cancer. Core-crosslinking by incorporating a disulfide bridge is a useful approach to improve the in vivo stability of polymeric micelles. This paper aims to investigate the effects of different crosslinking degrees on the anti tumor efficacy of micelles formed by poly(ethylene glycol methyl ether acrylate)b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA-PTX) block copolymer. Both 2D and 3D in vitro prostate tumor cell models were used to evaluate the uncrosslinked and crosslinked micelles. The cytotoxicity decreased with an increase in crosslinking degrees when tested with 2D cultured cells and all micelles remained less cytotoxic than free PTX. In the 3D prostate MCTS model, however, there was no statistical difference between the performance of uncrosslinked micelles and free PTX, whilst increasing crosslinking densities led to significantly relevant decreases in the antitumor efficacy of micelles. These results are contradictory to our previous research using an irreversible cross-linker (1,8-diaminooctane) to stabilize POEGMEA-b-PCEA-PTX conjugate micelles where it was shown that crosslinking accelerates and improves the effects of the micelles when compared to uncrosslinked micelles. Further studies are desired to investigate the underlying mechanisms of disulfide bonds when micelles are internalized into cells.

Keywords: Micelles; paclitaxel; crosslinking; cystamine; multicellular tumor spheroids

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Introduction Research into the use of polymeric nanoparticles as delivery vehicles for therapeutic agents has been a highly active field with many avenues of research. Nanoparticles are able to protect and deliver extremely hydrophobic and toxic drugs whilst allowing for virtually limitless control over the structure of the particle. These desirable traits have made nanoparticles a promising tool for cancer therapy1-3. The interest in using polymeric micelles as a potential delivery agent originates from its relatively simple shell-core structure. That is, the hydrophobic core allows for encapsulation of the therapeutic agent whilst the hydrophilic shell protects the contents from the external environment4-8. In particular, structures like micelles are able to passively target tumour sites due to both their nanoscale size and the enhanced permeability and retention (EPR) effect9. They are also able to avoid detection by the mononuclear phagocyte system (MPS)10. Micellar delivery of extremely potent drugs such as doxorubicin and paclitaxel are often the focus of current research due to the importance of such drugs in cancer therapy11, 12. Paclitaxel (PTX), for example, is a highly effective agent due to as it is able to inhibit the depolymerization of microtubules and in turn, cause apoptosis in the effected cell13. With an aqueous solubility of 1 µg/mL14, paclitaxel is currently delivered using Cremophor EL and ethanol, a delivery vehicle with its own host of adverse side effects15. Hence, micellar of paclitaxel have been previously explored using both physical16-19 and chemical20-23 encapsulation means to treat a range of cancers, including prostate cancer24. Chemical modification of micelles can also be easily achieved if factored into the initial design of the structure. Crosslinking, whether shell or core-crosslinking, is often used to enhance the stability of the micelle, especially against dissociation upon dilution or exposure to biological conditions25. In our previous work, we prepared PTX conjugated micelles from poly(ethylene glycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA-PTX) block copolymer and crosslinked the micelles with 1,8-diaminooctane (DAO)26. The core of the micelles were then irreversibly crosslinked using DAO to create amide bonds between the carboxylic acid groups on the polymer chains and free amine groups on the crosslinker. The crosslinking with DAO decreased the IC50 of un-crosslinked micelle in two-dimensionally (2D) cultured prostate tumor cells. It was more interesting, however, that crosslinking accelerated antitumor activities especially when compared with both uncrosslinked micelles and free PTX in a three-dimensional (3D) multicellular tumor spheroid (MCTS) model, which more accurately mimics the complexity found in in vivo tumors27-29. The enhancement in anti-tumor effects of the DAO cross-linked micelles was thought to be due to the increase in its stability. This enhancement allowed the micelles to penetrate LNCaP MCTS faster and deeper 26, 30. Crosslinking using disulfide compounds is an approach often used to introduce bonds which are reducible under the physiological conditions found within the cytoplasm of cancerous cells as well as aid in keeping drugs encapsulated within the core31. We hypothesize that the penetration and antitumor efficacy of the micelles can be influenced by crosslinking and changes of crosslinking degrees with reducible bonds. Therefore, the work presented in this paper is focused on developing PTX conjugated micellar delivery vehicles which feature different degrees of crosslinking with a reversible disulfide core-


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crosslinker for the treatment of prostate cancer. The micelles were then tested on both 2D and 3D cell culture models in an effort to demonstrate whether crosslinking density affected the overall efficacy of the micelle-drug formulation, especially when applied to a 3D macrostructure.



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Experimental Section Materials All materials were reagent grade and used as received, unless otherwise specified. 1,4-dioxane (>99%, Sigma-Aldrich), 2-(5-norborene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU, >99.9%, Chem-Impex International), 3-mercaptopropionic acid (>99%, Sigma-Aldrich), 5,5’-dithiobis(2-nitronezoic acid) (DTNB, >98%, Sigma-Aldrich), 9-aminoacridine hydrochloride monohydrate (>98%, Sigma-Aldrich), acetonitrile (ACN, >99.5%, Ajax Finechem), carboxyethyl acrylate (CEA, Sigma-Aldrich), cystamine dihydrochloride (CYS, 96%, Sigma-Aldrich) diisopropylethylamine (DIPEA, 99.9%, Sigma-Aldrich), fluorescein O-methacrylate (97%, SigmaAldrich), methanol (>98%, Ajax Finechem), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, BioXtra, Sigma-Aldrich), N,N-dimethylacetamide (DMAc, >99.9%, Sigma-Aldrich), N-hydroxysuccinimide (NHS, 98%, Sigma-Aldrich), paclitaxel (PTX, >99%, Dato Chemicals), oligo ethylene glycol methyl ether acrylate (OEGMEA, Mn = 480, Sigma-Aldrich), tris(2carboxyethyl)phosphine hydrochloride (TCEP, >98%, Sigma-Aldrich) and toluene (>99.5%, Ajax Finechem) were used without further purification. The RAFT agent, 332 benzylsulfanylthiocarbonylsufanylpropionic acid (BSPA), was synthesised previously . The initiator, 2,2’-Azobisisobutyronitrile (AIBN) was re-crystallized twice from methanol. MilliQ was produced internally and had a resistivity of 18.2 mΩ/cm.

Experimental Synthesis of BSPA-POEGMEA MacroRAFT OEGMEA (5.004 g; 1.04 × 10-2 mol) and BSPA (56.7 mg; 2.08 × 10-4 mol) were dissolved in toluene (10.4 mL) before AIBN was added (6.7 mg; 4.08 × 10-5 mol). The solution was purged with nitrogen for 30 min before the flask was sealed and immersed in an oilbath (60 °C, 75min). The polymerization was quenched by cooling the reaction solution in an icebath and exposing the solution to air. The polymer was obtained via dialysis against methanol (3× change) and then MilliQ water (2 d) before it was isolated via lyophilization. The conversion of POEGMEA, as determined by 1 H-NMR, was 33%. Synthesis of POEGMEA-b-PCEA copolymer The BSPA-POEGMEA macroRAFT agent (118.1 mg; 1.44 × 10-5 mol) and CEA (213.2 mg, 1.48 × 10-3 mol) were dissolved in 1,4-dioxane (1.95 mL) before AIBN (1.0 mg; 6.09 × 10-6) was added. Freezepump-thaw (3 cycles) was used to remove oxygen from the reaction solution before the flask was back-filled with nitrogen. The polymerization proceeded at 60 °C for 2 h, after which the reaction was quenched by cooling the mixture in an icebath and exposing the solution to air. The polymer was purified via dialysis against MilliQ water (4x change) and isolated via lyophilisation. The conversion of CEA, as determined by 1H-NMR, was 68%. Synthesis of Paclitaxel-polymer conjugate The POEGMEA-b-PCEA copolymer (62.8 mg; 3.49 × 10-6 mol) was dissolved in DMSO (1.8 mL). DIPEA (82.0 µL, 4.70 × 10-4 mol) and TNTU (23.8 mg; 2.05 × 10-4 mol) were added successively. The mixture stirred until the TNTU was completely dissolved (c.a. 5 min) before PTX (200 mL of 100 mg/mL stock solution in DMSO; 2.34 × 10-5 mol) was added. The flask was sealed and left to stir at 40 °C for 72 h.


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The paclitaxel-polymer conjugate was purified via dialysis (MWCO = 6000 - 8000) against acetonitrile (3 × solvent change), acetonitrile/MilliQ water (50 : 50; 3 × solvent change) and finally MilliQ water (5 × solvent change). After the final dialysis stage against MilliQ water, the purified conjugate (POEGMEA-b-PCEA-PTX) naturally formed micelles in solution. A portion was isolated via lypophilization and used for characterisation. The number of PTX moieties attached per polymer chain, as determined by 1H-NMR, was 3.6 units. Micellisation of paclitaxel-polymer conjugate Micellisation of the conjugate naturally occurred during the last dialysis stage in MilliQ water as outlined in the section above. Characterization of the micelles was carried out using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Concentration of the uncrosslinked polymer-conjugate micelles (CYS-UC) in solution was determined to be 3.2 mg/mL. Core-crosslinking with cystamine The micellar solution obtained after purification via dialysis in the above step was used without modification. Target crosslinking densities were 25, 50 and 75% of the available carboxylic acid groups present in the second block of the copolymer. To three separate vials of the micellar solution (1.5 mL/vial), 75 µL of the stock solutions of NHS (3.8 mg/mL), EDC (6.4 mg/mL) and cystamine (3.4 mg/mL) were added in the order listed with 5 min stirring after addition of each reagent. After the addition of cystamine, the samples were stirred gently for 24 h at room temperature. The samples were purified via dialysis (MWCO = 3 500; 5 × water change). The final concentrations of the samples after dialysis were determined via lyophilisation of a known volume of each solution. The concentrations determined were 2.65 mg/mL for 25% targeted crosslinking (CYS-L), 2.75 mg/mL for 50% targeted crosslinking (CYS−M) and 3.00 mg/mL for 75% targeted crosslinking (CYS−H). Determination of crosslinking content The micellar solution prepared in the previous step was used without modification. To 200 µL of each of the solutions of CYS-L, CYS-M and CYS-H, an aliquot of a 3 mg/mL solution of TCEP, in MilliQ water, was added (53 µL, 102 µL and 156 µL respectively). The solutions were incubated at 37 °C with shaking. After 3 hours, the solutions were transferred to a Micro Float-A-Lyzer device and dialysed against MilliQ water (MWCO = 3 500; 3 × water change) to remove TCEP. After dialysis, 30 µL of each micellar solution was added to 970 µL of PBS (pH = 7.4) and 50 µL of Ellman’s reagent (4 mg/mL of DTNB in PBS). After vigorous mixing, the absorbance of the solution was measured at 412 nm and thiol content determined by comparing to a standard curve made with 3mercaptopropanoic acid. 9-aminoacridine attachment to micelles as fluorescence labelling 9-aminoacridine hydrochloride monohydrate was initially desalted by dissolving 50 mg of the compound in a 50:50 mixture of water and ethyl acetate (20 mL). 500 mg of NaOH pellets were added to the mixture and stirred for 1 h at room temperature. The mixture was then extracted with DCM (3 × 40 mL) before the organic layer was dried over sodium sulfate and then removed under reduced pressure to yield a bright yellow powder.



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To a 500µL solution of each sample of micelles (c.a. 1.5mg/mL), 30 µL of EDC (2.1 mg/mL) and NHS (1.3 mg/mL) stock solutions (in MilliQ water) was added with stirring. To each solution, 300 µL of a 1mg suspension of 9-aminoacridine in MilliQ water was added and the mixtures stirred for 48hr at 40°C in an orbital shaker. Unreacted 9-aminoacridine was removed via dialysis (MWCO = 3 500, 10 × water change) against MilliQ water. In vitro evaluation of paclitaxel-polymer conjugate micelles In vitro cell expansion Human LNCaP cells (prostate carcinoma) were cultured in vitro for the evaluation of the paclitaxelcopolymer micelles. The cells were cultured in complete medium composed of RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 2.5 µg/mL Plasmocin as an antibiotic at 37 °C and 5 % CO2. Upon confluence, the cells were collected from tissue culture plastic surfaces with trypsin/EDTA treatment (3 min) before being seeded into new tissue culture vessels for evaluation. All in vitro testing was conducted on LNCaP cells which have been passaged less than 25 times. Intracellular micelle distribution via confocal microscopy 2 mL of LNCaP (0.5 × 105 cells per mL) cell suspension was seeded in a 35 mm Fluoro dish and cultured for 2 d. Fluorescent micelles (150 µg/mL) were added onto the cells and incubated for 1 hr. The cells were then washed three times with phosphate buffered saline (PBS, pH 7.4) and stained with 2.0 µg/mL Hoechst 33342 for 5 min followed by washing with PBS (3 ×). The cells were then stained with 100 nM LysoTracker for 1 min and rinsed once with PBS. The cells were mounted in 1mL Hank's Balanced Salt Solution (HBSS) and observed under the laser scanning confocal microscope. Sulforhodamine B assay (2D cell studies) for IC50 100 µL of LNCaP cell suspension was plated in a 96-well tissue culture plate at a density of 7 × 104 cells per mL. The cells were incubated for 24 h at 37 °C and 5% CO2 before the medium was removed from the plate. 100 µL of twice-concentrated RPMI-1640 medium was then added to each well (100 µL). For the control cells, the total volume of culture medium was increased to 200 µL by adding 100 µL sterile MilliQ water. The micelle solutions were sterilised by UV irradiation for 20 min in a biosafety cabinet before the solution was serially diluted by sterile MilliQ water at a ratio of 1: 1. The micellar solutions were then loaded into each well of the plate (100 µL). The plate was incubated with micelles for 3 days at 37 °C and 5 % CO2 for 72 h. The samples which were tested include free paclitaxel, uncrosslinked POEGMEA-b-PCEA-PTX conjugate micelles (CYS-UC) and cystamine-crosslinked micelles with different targeted crosslinked densities (CYS-L, CYS-M and CYS-H). The highest concentration of paclitaxel (equiv.) tested was 250 nM for free PTX and 5 µM for the micellar solutions. DMSO was used to dissolve free PTX at a concentration of 1 × 10-4 M and the appropriate solution was made by dilution with MilliQ water. The final working concentration of DMSO is 0.1 % in the cell culture medium. After 72 h, the cells were crosslinked by treatment with 10 % cold trichloroacetic acid (TCA) for 40 min at 4 °C. The cells were washed with water 5 times to remove the excess TCA. The cells were then


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stained with 0.4 wt% Sulforhodamine B (SRB) solution in 1 % acetic acid in dark for 20 min at room temperature. The plates were washed with 1 % acetic acid 5 times and air-dried. Then tris(hydroxymethyl)aminomethane (TRIS) buffer solution (200 µL) was added to each well to dissolve bound SRB. The plates were then read at 490 nm to record the absorbance of each well on a Multiskan Ascent plate reader and the data were plated and analysed with GraphPad Prism 6.0. Each micelle variant study was conducted in triplicate, where each study contained 6 parallel wells. The IC50 data is represented as mean ± standard deviation, where n = 3. A one way analysis of variance (ANOVA) was used to analyse the statistical difference among groups. Tukey's post hoc test was then used for multiple comparisons to reveal the difference between each group. Preparation of 3D multicellular tumour spheroid (MCTS) LNCaP cell suspension at a density of 1.5 × 105 cells/mL was used to prepare MCTS using a hanging drop method. The cell suspension was gently placed, dropwise (10 µL/drop), on to the inside surface of the lid of tissue culture dishes. Sterile PBS solution was added to the dish and the lid was placed back on the dish such that the cell droplets were suspended over the PBS solution inside the dish. The spheroids were cultured at 37 °C and 5% CO2 for 5 d. After 5 d, the spheroids were transferred to an U bottom 96 well plate with ultra-low attachment surface (Corning). Each well of the plate was supplemented with 200 µL RPMI-1640 media. The plate was then rotated on a slowly-moving 4way rotator for 24 h at the same incubation condition as that described for spheroid culture before the spheroids were used in subsequent tests. Intra-spheroid micelle distribution via confocal microscopy After the LNCaP spheroids were prepared as described above, the spheroids were incubated with fluorescent micelles (250 µg/mL) for 2 h before washing with PBS thrice. The spheroids were then mounted in PBS for confocal observation. Acid phosphatase (APH) assay For each well, 170 µL of media was gently taken out and replenished with 200 µL of fresh RPMI-1640 media. The spheroids were then incubated with PTX (free), CYS-UC, CYS-L, CYS-M or CYS-H micelles. The concentration of paclitaxel (equiv.) in the micelles was 10 µM and was the same across all samples. At day 7, the medium and micelles were replenished. The spheroid morphology was recorded under a phase contrast optical microscope. An APH assay was applied to determine the cell viability of the MCTS. Briefly, at each time point, the U bottom 96-well plates were centrifuged for 5 min at 500 g. The supernatant was carefully discarded and the spheroids were washed with PBS. The washing process was repeated two more times and the supernatant was discarded to a final volume of 100 µL. 100 µL of APH assay buffer was added to each well and the plates were incubated for 120 min at 37 °C and 5% CO2. The reaction was stopped by adding 10 µL of NaOH (1 M) and the absorption was read at 426 nm using a microplate reader (Ascent) within 10 min . The APH assay buffer was an aqueous solution of 2 mg/mL p-nitrophenyl phosphate, 0.1 M sodium acetate and 0.1 % (v/v) Triton X-100 (pH 4.8) where the p-nitrophenyl phosphate was added just before use. Eight spheroids were used for each sample tested at each time point. The data represent average ± standard deviation, n = 8. A two way analysis of variance (ANOVA) was used to analyse the statistical difference among groups. The Tukey's post hoc test was then used to reveal the difference between each group.



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Instrumental Analysis Nuclear magnetic resonance (1H-NMR) All NMR analysis were conducted on a Bruker Avance III HD instrument (1H, 600MHz) using a TCI cryoprobe. Deuterated dimethyl sulfoxide (DMSO-d6) was used as the solvent and peaks are assigned within each respective spectrum. All spectra were collected using a minimum of 8 scans and all chemical shifts are reported in ppm (δ). DMAc gel permeation chromatography (GPC) The GPC system for polymer characterization was a Shimadzu system consisting of a DGU-12A degasser, LC-10AT pump (1 mL/min), SIL-10AD automatic injector (50 µL), CTO-10A column oven (50 °C) and RID-10A refractive index detector (290 nm). Four Phenomenex columns in series were used for analyses (105, 104, 103 and 102 Å pore size, 5 μm particle size), where the mobile phase was DMAc (containing 0.05 w/v% BHT and 0.03 w/v% LiBr). Commerical polystyrene and polymethyl methacrylate standards were used for calibration, where molecular weight range of the standards was 200 – 106 g/mol. Each sample was dissolved in DMAc (ca. 4 - 5 mg/mL) before filtering through a 0.45 µm filter and then injection into the instrument. Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Dynamic light scattering (DLS) DLS measurements were conducted on a Bookhaven ZetaPlus Particle Sizer with a Dust-Cutoff value of 40. For size determination, polymer samples were prepared in MilliQ water at a concentration of 0.5 mg/mL and then filtered through a 0.45 µm filter to remove any dust particulates just before it sample measurement. For samples used to confirm crosslinking presence, the micelles were dissolved in DMSO (ca. 0.5 mg/mL) and measured without prior filtration. Each measurement was repeated in triplicate and averaged to give the listed values. Transmission electron microscopy (TEM) TEM images were obtained with a JEOL1400 instrument where the working beam voltage was 80100 kV. Samples were prepared by placing a drop of relevant micelle variant solution on a FORMVAR-coated copper grid. The solution was left for 30 min, under cover, to allow settling of particles on to the grid. Excess solution was removed using filter paper and the grids were air dried before being stained with uranyl acetate (3 % aqueous solution). The samples were exposed to the stain for 5 min before excess stain was removed and grids air dried before observation. Laser scanning confocal microscopy Micellar internalisation and penetration in spheroids were observed under a Zeiss LSM 780 laser scanning confocal microscope system. The system equipped with a Diode 405-30 laser, an argon laser and a DPSS 561-10 laser connected to a Zeiss Axio Observer.Z1 inverted microscope. The ZEN2012 imaging software (Zeiss) was used for image acquisition and processing. The 2D cultured cells were observed with a 100 × 1.4 NA oil objective and the spheroid was observed with a 20 × 0.8 NA air objective.


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Results and Discussion Conjugation of paclitaxel to POEGMEA-b-PCEA Table 1: Conversion, calculated molecular weight and polydispersity of POEGMEA, POEGMEA-b-PCEA and POEGMEA-bPCEA-PTX conjugate

Compound

Conversion (%)a

Mn (g/mol)b

Ðc

POEGMEA17 macroRAFT

33

8 200

1.21

POEGMEA17-b-PCEA68

68

18 000

1.09

POEGMEA17-b-PCEA68-PTX4 conjugate a Monomer conversion b Determined via 1H-NMR c Determined via GPC

-

21 000

1.18

Table 1 provides a summary of the conversion and polydispersity of the initial POEGMEAmacroRAFT, the chain extended block copolymer as well as the final POEGMEA-b-PCEA-PTX conjugate. The conjugation of paclitaxel to the POEGMEA-b-PEA block polymer was achieved using solid phase peptide reagents as outlined previously26. The successful conjugation was confirmed using 1H-NMR (Figure 1) and GPC (data not shown). The amount of paclitaxel units attached per polymer chain was calculated (via 1H-NMR) to be approximately 4 units per chain or 15 wt% of polymer-paclitaxel conjugate, where attachment at the carboxylic acid end of the chain is also possible, but unable to be clearly shown via characterization techniques. The low polydispersity of the polymers after each step highlights the controlled nature of the polymerizations. At ~4 units per polymer chain, the conjugation efficiency of paclitaxel achieved was 53 % (relative to total paclitaxel added) and is again comparable to efficiencies achieved by traditional carbodiimide reagents 20-22, 33, 34 without the need for anhydrous and/or inert conditions.

1

Figure 1: H-NMR assignment of poly(oligo ethylene glycol methyl ether acrylate)-block-poly(carboxyethyl acrylate)paclitaxel (POEGMEA-b-PCEA-PTX) conjugate

Crosslinking of POEGMEA-b-PCEA-PTX conjugate micelles with cystamine

The micelles naturally formed from this particular polymer-drug conjugate system were approximately 160 – 240 nm in size when in solution (Table 2). Crosslinking of the micelles was then



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achieved by introducing cystamine to the micelle solution in different amounts and using EDC to facilitate the amide formation between the amines on cystamine and free carboxylic acid groups on the PCEA block. After 24h, unconsumed reagents were removed via dialysis to afford the crosslinked species. Successful crosslinking was confirmed via DLS of the micelles in DMSO (a good solvent for both blocks) and TEM. Table 2: Sizes and distribution of uncrosslinked and crosslinked micelles in water as determined by DLS

Micelle Samplea

Size (nm)

PDI

CYS-UC

161.0

0.133

CYS-L

235.5

0.319

CYS-M

217.2

0.221

CYS-H 182.3 0.238 a Where CYS-UC represents uncrosslinked micelles, CYS-L, CYS-M and CYS-H are micelles crosslinked with 25%, 50% and 75% (theo.) cystamine, respectively.

The size intensity distribution of the micelles in water and resulting PDI are listed in Table 2. The integrity of the particles was retained when the measurements were repeated using DMSO as a solvent (data not shown). Although all particles increased in size to ca. 250 – 300nm, which can be attributed to swelling of the core, all crosslinked samples remained intact, demonstrating the crosslinking success. TEM images of the four micelles species are shown in Figure 2 where all species are approximately 100nm. The disagreement with DLS sizes was expected in this case as shrinkage in micelle sizes to ~50% of the hydrated size was seen previously26. The shrinkage is thought to be typical with this particular block copolymer system due to the highly branched nature of the shell POEGMEA block.

Figure 2: TEM images of the four micelle variants (A) CYS-UC micelles, with 9-aminoacridine attached to the core, (B) CYS-L, (C) CYS-M and (D) CYS-H. Scale bar = 200µ µm. Samples were stained with uranyl acetate.


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The crosslinking content of each of the micelle variants was determined by using Ellman’s reagent after digesting the disulfide bonds with TCEP. The absorbance of each of the micelle variants was read at 412nm and compared to a standard curve prepared using 3-mercaptopropionic acid to obtain the concentration of thiols in each sample. The ratio of thiols per polymer chain, calculated crosslinking density and cystamine conjugation efficiency is outlined in Table 3. From the table it can be seen that the conjugation efficiency of cystamine decreases with increasing amounts of cystamine, pointing to a possible plateauing effect. That is, there may be a theoretical upper limit to how many cystamine units can effectively penetrate the micelle core and then attach to two separate polymer chains. Although increasing the cystamine amount in the case of CYS-H did result in more cystamine conjugation, the increase was not as pronounced as that seen between CYS-L and CYS-M (which had the same increase in added cystamine). Table 3: Thiol content per polymer chain in CYS-L, CYS-M and CYS-H crosslinked micelles

Micelle Type

Thiols/polymer chain

CYS-L

9

Calculated crosslinking Cystamine conjugation density (%) efficiency (%) 14% 56

CYS-M

16

25%

50

CYS-H

19

30%

40

However, it is also possible that the lower than expected conjugation efficiency of CYS-M and CYS-H could be due to the reformation of disulfide bonds between the free thiol groups before the reaction with Ellman’s reagent could occur. Attempts were made to verify the results in Table 3 using a maleimide-based pyrene compound, which have previously been demonstrated to react with free thiols on polymer chains35Supprossad, but difficulty in obtaining a usable standard curve was not possible. It would appear that the nature of the conjugated compound onto the maleimide group drastically alters the fluorescence profile of the final species (further information can be found in Supporting Information). Thus, the results presented in Table 3 was taken to be indicative disulfide content achieved and 30% crosslinking was taken to be approaching the upper crosslinking threshold possible with this system. Uptake of micelles in 2D cultured cells and MCTS structures

Successful uptake of the micelles into cells arranged in both 2D and 3D structures was demonstrated using laser scanning confocal microscopy with 9-aminoacridine as the fluorescent agent. Uptake into the 2D cultured cells can be seen in Figure 3 where the micelles are endocytosed into the lysosomes of the cells. This is indicated via the overlap between the micelle signal (yellow) and lysosome signal (red) in the merged images. In addition, it can also be seen that the uptake of micelles decreased with an increase in crosslinking degree. Possible quenching of the 9-aminoacridine signal inside the micelle was tested via fluorescence measurements of CYS-M sample and it was demonstrated that no quenching occurred (data shown in SI).



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Figure 3: Penetration of POEGMEA-b-PCEA-PTX conjuguate micelles (CYS-UC, CYS-L, CYS-M, CYS-H) into LNCaP cells (2D culture) as determined by laser scanning confocal microscopy (scale bars = 20 μm). Micelles were labelled with 9aminoacridins and lysosomes labelled with LysoTracker.

Micelles can also penetrate into the MCTS structure, as seen in Figure 4A. From the fluorescence profile of the spheroid (Figure 4B, taken at the same depth as Figure 4A and normalized within each sample against the highest fluorescence intensity recorded), it can be seen that crosslinked micelles appear to penetrate more deeply than that of the uncrosslinked micelles. For the uncrosslinked micelles, significant penetration is seen at the edges of the spheroid, but fluorescence intensity decreases rapidly at a distance of approximately 100 µm from the edge of the spheroid. Conversely, for the CYS-L micelles, micelle penetration is stable and sustained for the outmost 200 µm and only starts to decrease rapidly thereafter. The profiles of CYS-M and CYS-H show that penetration of the micelles was well-sustained when compared to CYS-UC. Whilst the uncrosslinked micelles suffered from decreased penetration in the centre, with intensity values dropping to ca. 0.26, crosslinked micelles CYS-L, CYS-M and CYS-H had minimum intensities of 0.30, 0.39 and 0.43, respectively. Thus it can be seen that crosslinking micelles improves penetration of the particles.


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Figure 4: (A) Penetration of POEGMEA-b-PCEA-PTX conjugate micelles (CYS-UC, CYS-L, CYS-M, CYS-H) into LNCaP spheroids as determined by confocal microscopy at a depth of 90 μm into the MCTS (scale bars =50 μm) and (B) fluorescence profile of a horizontal slice of the LNCaP spheroid taken at a depth of 90 μm.

2D Cytotoxicity on LNCaP Cells

Despite the relatively small difference in crosslinking density as calculated above, a marked difference in cytotoxic effect can be clearly be seen via the 2D cytotoxicity evaluations (Figure 5) on prostate cancer cells with only CYS-L and CYS-M showing no significant difference from each other (Figure 6). Although none of the four micelle samples can match the toxicity of free paclitaxel (micromolar range compared to nanomolar toxicity), the micelles also performed demonstrably different from each other in general where uncrosslinked micelles performed the best. The performance of CYS-UC could be due to its smaller size and thus, higher uptake. Other micellar systems which employ disulfide containing compounds have also demonstrated lower micelle toxicities after crosslinking36. In the case of this work, the core becomes especially hydrophobic after crosslinking due to the presence of paclitaxel, which could affect the diffusion of intracellular reductive agents like glutathione. It is conceivable that higher crosslinking density equates slower micelle dissociation, which in turn leads to higher IC50 values (listed in Table 4). It is also interesting to note that despite the higher crosslinking density between CYS-L and CYS-M, there was no significant difference between their IC50 values. Indeed, if only the mean was taken into account, there is actually a slight decrease in IC50 value where CYS-M is statistically more similar to CYS-UC than CYS-L and it is unclear what is causing this phenomenon. As all other micelle groups CYS-UC, CYS-L and CYS-M have higher toxicities than CYS-H, it would be expected that CYS-M also follow this general trend and fall between CYS-L and CYS-H. As it does not it could be that there is an optimum crosslinking density when using cystamine as a reversible crosslinker and that that density falls in the range of CYS-L and CYS-M, namely approximately 25% or lower crosslinking density, especially when the core is naturally extremely hydrophobic.



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Figure 5: Cytotoxicity curves of free paclitaxel and PTX conjugated micelles on 2D cultured LNCaP cells.

Figure 6: IC50 values of the micelle samples CYS-UC, CYS-L, CYS-M and CYS-H as tested on 2D cultured LNCaP cells. Data represents means ± SD, n = 3; *, significant different, P < 0.05; **, significant different, P CYS-L > CYS-M > CYS-H, where CYSUC performs statistically better than all other micelles variants except CYS-L. At this point in the testing cycle, uncrosslinked micelles are the most effective against the prostate cancer MCTS even when added at the same paclitaxel concentration as the free paclitaxel. At day 7, there is also a statistical difference between CYS-L and CYS-M, with CYS-L performing better than its higher crosslinked counterpart. This contradicts the idea obtained from 2D testing, which suggests that CYSM may have been the optimum crosslinking density and instead implies that lower crosslinking results in higher efficacy. After 14 days of treatment, CYS-UC continues to demonstrate the greatest inhibition effect, with free paclitaxel being the second most effective. Although CYS-L, CYS-M and CYS-H exhibit decreasing inhibition ability in that order, the difference is less pronounced by day 14. Indeed there is almost negligible difference between CYS-L and CYS-M, a long-term result reflected by their IC50 values, but with no indication that CYS-M would be more effective than CYS-L despite the lower IC50 concentration. The most interesting effect to consider, however, is that there remains a statistically relevant difference between uncrosslinked micelles and both CYS-M and CYS-H, even at day 14. That is, even 25% crosslinking density of the micelles will negatively affect the performance of the micelles. Although not statistically relevant, CYS-L did appear to perform worse than CYS-UC in these tests, adding further weight to the idea that high crosslinking density using disulfide agents may adversely affect micelle performance. Optical visualization of the MCTS during the 14 day testing can be seen in Figure 8.

Figure 7: Cell viability of LNCaP MCTSs after 7 and 14 days of treatment with POEGMEA-b-PCEA-PTX conjugate micelles (with various degrees of crosslinking) and free paclitaxel. Data represent means ± SD, n = 8; *, significant difference, P < 0.05; **, significant difference, P < 0.01; ***, significant difference, P < 0.001.



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Figure 8: Effect of POEGMEA-b-PCEA-PTX conjugate micelles (CYS-UC, CYS-L, CYS-M and CYS-H) on the morphology of LNCaP spheroids when compared with free paclitaxel (scale bar = 300 μm)

The overall trend does suggest that if a disulfide reversible crosslinker is used for this system, it is most advantageous to keep the crosslinking density at a minimum despite the importance of micellar stability on extending circulation time. That is, crosslinking density does appear to have an overall effect on micellar efficacy and that such considerations must be made when designing a synthetic nanocarrier system. Another point worthy of notice is that the nature of the crosslinking agent may play a part in the overall cytotoxicity of the micelles. Previously26, we have shown that irreversibly crosslinking the cores of the micelle actually increased the toxicity of the uncrosslinked micelle. That is, the use of the diamino crosslinker 1,8-diaminooctane, produces micelles which were more toxic than its uncrosslinked counterpart both in 2D and 3D cellular models, a result which would appear to contradict the results shown here. As it has been seen before that crosslinking with such an irreversible reagent does indeed produce more toxic micelles37, it may be that the actual crosslinking agent itself plays a role other than stabilising the micelle structure. Thus, the effects of crosslinking agents and their reversible (or irreversible) nature is a topic which will require further investigation. In this work, paclitaxel was conjugated to POEGMEA-b-PCEA and the uncrosslinked micelles were formed after dialysis of the polymer against water. Crosslinking using cystamine improved the stability of micelles. Surprisingly, contrary from our previous work using DAO as an inreversible crosslinker, a degradable cystamine crosslinker increased the IC50 values of all micelle variants against 2D cultured LNCaP cells. A higher crosslinking degree resulted in a higher IC50. All micelle


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variants inhibited MCTS growth and reduced the viability of the spheroids after 14 days of treatment. CYS-UC performed better than free PTX throughout the testing period although the difference was not significant after 14 days. In addition, the inhibition effect decreased with an increase in crosslinking degree. Hence, there is a demand to find the optimum balance between micelle stability and micelle performance when cystamine is incorporated as a reversible crosslinker as its density appears to play an important part on overall micelle performance.

Associated Content Supporting Information: Fluorescence quenching experiment; Analysis of thiol content using N-(1pyrenyl)maleimide. The Supporting Information is available free of charge on the website.

Acknowledgement The authors would like to acknowledge the Australian Research Council (ARC) for funding

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Stabilization of Paclitaxel-Conjugated Micelles by Cross-Linking with

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